Erythrocytes (red blood cells or RBCs), leukocytes (white blood cells), and platelets comprise the cellular constituents of mammalian blood. Healthy nonpathological erythrocytes are flexible, biconcave disks that lack both nuclei and mitochondria. Erythrocytes contain an allosteric, iron-containing metalloprotein called hemoglobin (Hb), that binds oxygen and carbon dioxide and enables erythrocytes to transport these gases in the blood. Hemoglobin consists of four subunits, each containing a nonprotein heme group surrounded by the globin protein portions of the molecule. Each heme group contains an iron (Fe) atom held in the center of a heterocyclic porphyrin ring. Like all heme-containing proteins, Hb absorbs electromagnetic radiation in the visible and near ultraviolet (UV) portions of the spectrum. This characteristic allows Hb to be detected and quantified using spectrophotometric methods.
The iron atom of the Hb contained within erythrocytes exists in either oxidation state Fe2+ or Fe3+. Iron in the Fe2+ state binds oxygen and forms oxyhemoglobin (Oxy-Hb) in the pulmonary capillaries of the lungs. Other gas molecules compete with oxygen for the heme binding site and form heme derivatives. Notably, carbon monoxide binds the heme iron in the Fe2+ state and forms carboxyhemoglobin (CO-Hb), reducing the Hb available for oxygen transport. Deoxyhemoglobin (Doxy-Hb), the reduced (Fe2+) state of Hb with no bound oxygen, is formed after the oxygen is released to the tissues. Oxidation to the Fe3+ state converts hemoglobin to methemoglobin (Met-Hb), which cannot bind oxygen. Oxy-Hb and Deoxy-Hb comprise about 90% of the total Hb in blood.
Although hemoglobin exists largely within the erythrocytes, healthy blood plasma contains a small amount of cell-free Hb, usually less than 0.05% of total hemoglobin. However, the cell-free Hb levels sometimes increase significantly in response to pathological hemolytic conditions such as sickle cell anemia, paroxymal hemoglobinuria, acute autoimmune hemolytic anemia, transfusion reactions due to blood group incompatibilities and faulty intracardiac valvular prostheses. Medical procedures requiring manipulation of the blood such as dialysis, or cardiac bypass procedure can also cause hemolysis. It is therefore sometimes medically important to have a rapid and inexpensive method to measure plasma hemoglobin.
Healthy erythrocytes are biconcave cells that have sufficiently elastic membranes to allow the erythrocyte to elongate and pass through the capillaries within the circulatory system. Blood for transfusion is commonly stored for several weeks under refrigeration with an anticoagulant such as heparin, citrate or an anticoagulant and preservative such as CPDA-1. During storage, erythrocyte membranes lose their elasticity and become fragile and prone to rupture during handling or after it the blood given to a patient. It is therefore sometimes desirable to determine erythrocyte membrane elasticity before using the stored blood to transfuse a patient.
In medical practice, the plasma cell-free Hb level is determined by obtaining a sample of blood and adding an anticoagulant, usually heparin. The cellular fraction is precipitated by centrifugation and the plasma is removed. An estimate of cellular erythrocyte hemoglobin can be obtained by determining the hematocrit, the packed red cell volume. The plasma is analyzed for cell-free hemoglobin content either by enzymatic or spectrophotometric methods. The enzymatic methods rely on using the pseudo-peroxidase activity of Hb to act upon a substrate such as benzedine, parpminobenzoic acid or tetramethylbenzidine to produce readily quantifiable reaction products.
Several spectrophotometric methods to determine plasma concentrations of cell-free Hb have been described. One such method requires derivatizing most forms of hemoglobin with Drabkin's reagent and forming cyanomethemoglobin, which has an absorbance peak at 540 nm wavelength. Other methods allow direct measurement of plasma hemoglobin concentration by scanning spectroscopic methods using visible and near infrared portions of the electromagnetic spectrum. Any of these methods can also be used to evaluate the relative amount of ruptured erythrocytes thus providing an indirect measure of erythrocyte membrane fragility, based on cell-free Hb concentration in plasma. However not all changes in membrane fragility result in erythrocyte rupture and increased cell-free Hb. In addition, all of these methods share the limitation that centrifugation to remove the erythrocytes is required before analysis can take place, and thus, require access to laboratory equipment and do not provide the speed necessary for rapid analysis.
It would be desirable, to provide a system and method for measuring the concentration of Hb in bodily fluids such as blood, plasma, serum, and urine without having to remove erythrocytes and other cellular components, and without requiring that chemical or enzymatic reactions be conducted. Such a system and method would overcome many of the limitations and disadvantages inherent in those described above.